Translesion DNA Replication Research Articles

Quantifying the energetic contributions of desolvation and π-electron density during translesion DNA synthesis

This report examines the molecular mechanism by which high-fidelity DNA polymerases select nucleotides during the replication of an abasic site, a non-instructional DNA lesion. This was accomplished by synthesizing several unique 5-substituted indolyl 2′-deoxyribose triphosphates and defining their kinetic parameters for incorporation opposite an abasic site to interrogate the contributions of π-electron density and solvation energies. In general, the Kd, app values for hydrophobic non-natural nucleotides are ∼10-fold lower than those measured for isosteric hydrophilic analogs. In addition, kpol values for nucleotides that contain less π-electron densities are slower than isosteric analogs possessing higher degrees of π-electron density. The differences in kinetic parameters were used to quantify the energetic contributions of desolvation and π-electron density on nucleotide binding and polymerization rate constant. We demonstrate that analogs lacking hydrogen-bonding capabilities act as chain terminators of translesion DNA replication while analogs with hydrogen bonding functional groups are extended when paired opposite an abasic site. Collectively, the data indicate that the efficiency of nucleotide incorporation opposite an abasic site is controlled by energies associated with nucleobase desolvation and π-electron stacking interactions whereas elongation beyond the lesion is achieved through a combination of base-stacking and hydrogen-bonding interactions.

Translesion DNA replication proteins as molecular targets for cancer prevention

Mutations in DNA are generally considered to have an etiologic role in the development of cancer. If so, it follows that reducing the frequency of such mutations will reduce the incidence of cancer induced by mutagens. Recent advances in elucidating the molecular mechanisms of carcinogen-induced mutagenesis indicate that replication of DNA templates that contain replication-blocking adducts is accomplished with error-prone DNA polymerases. These polymerases have relaxed base-pairing requirements, and can insert bases across from adducted templates, but with potentially mutagenic consequences. In principle, these proteins present new and attractive molecular targets to reduce mutagenesis. If this can be done in vivo without increasing cytotoxic responses to carcinogens, then novel chemopreventive strategies can be designed to reduce the risk of cancer in exposed populations prior to the appearance of disease symptoms.

Dual roles of glycosyl torsion angle conformation and stereochemical configuration in butadiene oxide-derived N1 beta-hydroxyalkyl deoxyinosine adducts: a structural perspective.

The solution structure of the N1-[1-hydroxy-3-buten-2(R)-yl]-2'-deoxyinosine adduct arising from the alkylation of adenine N1 by butadiene epoxide (BDO), followed by deamination to deoxyinosine, was determined in the oligodeoxynucleotide 5'-d(CGGACXAGAAG)-3'.5'-d(CTTCTTGTCCG)-3'. This oligodeoxynucleotide contained the BDO adduct at the second position of codon 61 of the human N-ras protooncogene (underlined) and was named the ras61 R-N1-BDO-(61,2) adduct. 1H NMR revealed a weak C5 H1' to X6 H8 nuclear Overhauser effects (NOE), followed by an intense X6 H8 to X6 H1' NOE. Simultaneously, the X6 H8 to X6 H3' NOE was weak. The resonances arising from the T16 and T17 imino protons were not observed. 1H NOEs between the butadiene moiety and the DNA positioned the adduct in the major groove. Structural refinement based upon a total of 394 NOE-derived distance restraints and 151 torsion angle restraints yielded a structure in which the modified deoxyinosine was in the syn conformation about the glycosyl bond, with a glycosyl bond angle of 83 degrees , and T17, the complementary nucleotide, was stacked into the helix but not hydrogen bonded with the adducted inosine. The refined structure provides a plausible hypothesis as to why these N1 deoxyinosine adducts strongly code for the incorporation of dCTP during trans lesion DNA replication, irrespective of stereochemistry, both in Escherichia coli [Rodriguez, D. A., Kowalczyk, A., Ward, J. B. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2001) Environ. Mol. Mutagen. 38, 292-296] and in mammalian cells [Kanuri, M., Nechev, L. N., Tamura, P. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2002) Chem. Res. Toxicol. 15, 1572-1580]. Rotation of the N1 deoxyinosine adduct into the syn conformation may facilitate incorporation of dCTP via Hoogsteen type templating with deoxyinosine, generating A to G mutations. However, conformational differences between the R- and the S-N1-BDO-(61,2) adducts, involving the positioning of the butenyl moiety in the major groove of DNA, suggest that adduct stereochemistry plays a secondary role in modulating the biological response to these adducts.

Structure of an Oligodeoxynucleotide Containing a Butadiene Oxide-Derived N1 Beta-Hydroxyalkyl Deoxyinosine Adduct in the Human N-ras Codon 61 Sequence

The solution structure of the N1-(1-hydroxy-3-buten-2(S)-yl)-2'-deoxyinosine adduct arising from the alkylation of adenine N1 by butadiene epoxide (BDO), followed by deamination to deoxyinosine, was determined, in the oligodeoxynucleotide d(CGGACXAGAAG).d(CTTCTCGTCCG). This oligodeoxynucleotide contained the BDO adduct at the second position of codon 61 of the human N-ras protooncogene, and was named the ras61 S-N1-BDO-(61,2) adduct. (1)H NMR revealed a weak C(5) H1' to X(6) H8 NOE, followed by an intense X(6) H8 to X(6) H1' NOE. Simultaneously, the X(6) H8 to X(6) H3' NOE was weak. The resonance arising from the T(17) imino proton was not observed. (1)H NOEs between the butadiene moiety and the DNA positioned the adduct in the major groove. Structural refinement based upon a total of 364 NOE-derived distance restraints yielded a structure in which the modified deoxyinosine was in the high syn conformation about the glycosyl bond, and T(17), the complementary nucleotide, was stacked into the helix, but not hydrogen bonded with the adducted inosine. The refined structure provided a plausible hypothesis as to why this N1 deoxyinosine adduct strongly coded for the incorporation of dCTP during trans lesion DNA replication, both in Escherichia coli [Rodriguez, D. A., Kowalczyk, A., Ward, J. B. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2001) Environ. Mol. Mutagen. 38, 292-296], and in mammalian cells [Kanuri, M., Nechev, L. N., Tamura, P. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2002) Chem. Res. Toxicol. 15, 1572-1580]. Rotation of the N1 deoxyinosine adduct into the high syn conformation may facilitate incorporation of dCTP via Hoogsteen-type templating with deoxyinosine, thus generating A-to-G mutations.

Enzymatic switching for efficient and accurate translesion DNA replication.

When cyclobutane pyrimidine dimers stall DNA replication by DNA polymerase (Pol) delta or epsilon, a switch occurs to allow translesion synthesis by DNA polymerase eta, followed by another switch that allows normal replication to resume. In the present study, we investigate these switches using Saccharomyces cerevisiae Pol delta, Pol epsilon and Pol eta and a series of matched and mismatched primer templates that mimic each incorporation needed to completely bypass a cis-syn thymine-thymine (TT) dimer. We report a complementary pattern of substrate use indicating that enzymatic switching involving localized translesion synthesis by Pol eta and mismatch excision and polymerization by a major replicative polymerase can account for the efficient and accurate dimer bypass known to suppress sunlight-induced mutagenesis and skin cancer.

Inactivation of the 3′-5′ Exonuclease of the Replicative T4 DNA Polymerase Allows Translesion DNA Synthesis at an Abasic Site

Here, we have investigated the consequences of the loss of proof-reading exonuclease function on the ability of the replicative T4 DNA polymerase (gp43) to elongate past a single abasic site located on model DNA substrates. Our results show that wild-type T4 DNA polymerase stopped at the base preceding the lesion on two linear substrates having different sequences, whereas the gp43 D219A exonuclease-deficient mutant was capable of efficient bypass when replicating the same substrates. The structure of the DNA template did not influence the behavior of the exonuclease-proficient or deficient T4 DNA polymerases. In fact, when replicating a damaged “minicircle” DNA substrate constructed by circularizing one of the linear DNA, elongation by wild-type enzyme was still completely blocked by the abasic site, while the D219A mutant was capable of bypass. During DNA replication, the T4 DNA polymerase associates with accessory factors whose combined action increases the polymerase-binding capacity and processivity, and could modulate the behavior of the enzyme towards an abasic site. We thus performed experiments measuring the ability of wild-type and exonuclease-deficient T4 DNA polymerases, in conjunction with these replicative accessory proteins, to perform translesion DNA replication on linear or circular damaged DNA substrates. We found no evidence of either stimulation or inhibition of the bypass activities of the wild-type and exonuclease-deficient forms of T4 DNA polymerase following addition of the accessory factors, indicating that the presence or absence of the proof-reading activity is the major determinant in dictating translesion synthesis of an abasic site by T4 DNA polymerase.

Evaluating the contributions of desolvation and base-stacking during translesion DNA synthesis.

DNA polymerases catalyze the insertion of a nucleoside triphosphate into the growing polymer chain using the template strand as a guide. Numerous factors such as hydrogen bonding interactions, base-stacking contributions, and desolvation play important roles in controlling the efficiency and fidelity of this process. We previously demonstrated that 5-nitro-indolyl-2'-deoxyriboside triphosphate, a non-natural nucleobase with enhanced base-stacking properties, was more efficiently inserted opposite a non-templating DNA lesion compared to natural templating nucleobases (E. Z. Reineks and A. J. Berdis, Biochemistry, 2004, 43, 393-404). The catalytic enhancement was proposed to reflect increased base-stacking interactions of the non-natural nucleobase with the polymerase and DNA. However, the effects of desolvation could not be unambiguously refuted. To further address the contributions of base stacking and desolvation during translesion DNA replication, we synthesized indolyl-2'-deoxyriboside triphosphate, a nucleobase devoid of nitro groups, and measured its efficiency of enzymatic insertion into modified and unmodified DNA. Removal of the nitro group reduces the catalytic efficiency for insertion opposite an abasic site by 3600-fold. This results from a large decrease in the rate of polymerization (similar 450-fold) coupled with a modest decrease in binding affinity (similar 8-fold). Since both non-natural nucleobases show the same degree of hydrophobicity, we attribute this reduction to the loss of base-stacking contributions rather than desolvation capabilities. Indolyl-2'-deoxyriboside triphosphate can also be inserted opposite natural nucleobases. Surprisingly, the catalytic efficiency for insertion is nearly identical to that measured for insertion opposite an abasic site. These data are discussed within the context of pi-electron interactions of the incoming nucleobase with the polymerase:DNA complex. Despite this lack of insertion selectivity, the polymerase is unable to extend beyond the non-natural nucleobase. This result indicates that indolyl-2'-deoxyriboside triphosphate acts as an indiscriminate chain terminator of DNA synthesis that may have unique therapeutic applications.

Ribozyme-mediated REV1 inhibition reduces the frequency of UV-induced mutations in the human HPRT gene.

In yeast, mutations induced by UV radiation are dependent on the function of the Rev1 gene product, a Y-family DNA polymerase that assists in translesion replication with potentially mutagenic consequences. Human REV1 has been cloned, but its role in mutagenesis and carcinogenesis remains obscure. To examine the role of REV1 in UV mutagenesis in human cells and to evaluate its potential as a therapeutic target to prevent such mutations, we developed a ribozyme that cleaves human REV1 mRNA in vitro. Stable expression of the ribozyme in human cells reduced the target REV1 mRNA up to 90%. We examined the cytotoxic and mutagenic response to UV of seven independent clones that had reduced levels of endogenous REV1 mRNA. In each case, the clonogenic survival after UV was not different from that of the parental cell strains. In contrast, the UV-induced mutant frequencies at the endogenous HPRT locus were reduced up to 75% in cells with reduced levels of REV1 mRNA. The data support the idea that targeting the mutagenic translesion DNA replication pathway can greatly reduce the frequency of induced mutations.

Evaluating the Effects of Enhanced Processivity and Metal Ions on Translesion DNA Replication Catalyzed by the Bacteriophage T4 DNA Polymerase

The fidelity of DNA replication is achieved in a multiplicative process encompassing nucleobase selection and insertion, removal of misinserted nucleotides by exonuclease activity, and enzyme dissociation from primer/templates that are misaligned due to mispairing. In this study, we have evaluated the effect of altering these kinetic processes on the dynamics of translesion DNA replication using the bacteriophage T4 replication apparatus as a model system. The effect of enhancing the processivity of the T4 DNA polymerase, gp43, on translesion DNA replication was evaluated using a defined in vitro assay system. While the T4 replicase (gp43 in complex with gp45) can perform efficient, processive replication using unmodified DNA, the T4 replicase cannot extend beyond an abasic site. This indicates that enhancing the processivity of gp43 does not increase unambiguously its ability to perform translesion DNA replication. Surprisingly, the replicase composed of an exonuclease-deficient mutant of gp43 was unable to extend beyond the abasic DNA lesion, thus indicating that molecular processes involved in DNA polymerization activity play the predominant role in preventing extension beyond the non-coding DNA lesion. Although neither T4 replicase complex could extend beyond the lesion, there were measurable differences in the stability of each complex at the DNA lesion. Specifically, the exonuclease-deficient replicase dissociates at a rate constant, k off, of 1.1 s −1 while the wild-type replicase remains more stably associated at the site of DNA damage by virtue of a slower measured rate constant ( k off 0.009 s −1). The increased lifetime of the wild-type replicase suggests that idle turnover, the partitioning of the replicase from its polymerase to its exonuclease active site, may play an important role in maintaining fidelity. Further attempts to perturb the fidelity of the T4 replicase by substituting Mn 2+ for Mg 2+ did not significantly enhance DNA synthesis beyond the abasic DNA lesion. The results of these studies are interpreted with respect to current structural information of gp43 alone and complexed with gp45.

Manganese substantially alters the dynamics of translesion DNA synthesis.

The effect of metal ion substitution on the dynamics of translesion DNA synthesis catalyzed by the bacteriophage T4 DNA polymerase was quantitatively evaluated through steady-state and transient kinetic techniques. Substitution of Mn(2+) for Mg(2+) enhances the steady-state rate of dNMP misinsertion opposite an abasic site by 11-34-fold. At the molecular level, the enhancement in translesion DNA synthesis reflects a substantial increase in the rate of the conformational change preceding phosphoryl transfer for all dNTPs that were tested. This is best illustrated by the biphasic pre-steady-state time course of dAMP insertion opposite an abasic site which indicates that a step after chemistry is rate-limiting for steady-state enzyme turnover. Furthermore, the k(pol) value of 40 s(-1) measured under single-turnover reaction conditions is 20-fold greater than the k(cat) value of 2 s(-1) measured for steady-state enzyme turnover. Finally, the low elemental effect ( approximately 2.4-fold reduction in k(pol)) measured by substituting the alpha-thiotriphosphate analogue for dATP further argues that chemistry is not rate-limiting. In contrast to the biphasic insertion of dAMP, pre-steady-state time courses for the insertion of dCMP, dGMP, or dTMP opposite an abasic site were linear. Nearly identical k(pol) values ( approximately 1 s(-1)) were measured for the insertion of dCMP, dGMP, and dTMP opposite the abasic site using single-turnover conditions. However, the large elemental effects of 27 and 70 measured by substituting the alpha-thiotriphosphate analogues for dCTP and dGTP, respectively, suggest that phosphoryl transfer may be the rate-limiting step for their insertion opposite the abasic site. Various models are discussed in an attempt to explain the effect of metal ion substitution on the dynamics of translesion DNA replication.

UmuDC-mediated cold sensitivity is a manifestation of functions of the UmuD(2)C complex involved in a DNA damage checkpoint control.

The umuDC genes are part of the Escherichia coli SOS response, and their expression is induced as a consequence of DNA damage. After induction, they help to promote cell survival via two temporally separate pathways. First, UmuD and UmuC together participate in a cell cycle checkpoint control; second, UmuD'(2)C enables translesion DNA replication over any remaining unrepaired or irreparable lesions in the DNA. Furthermore, elevated expression of the umuDC gene products leads to a cold-sensitive growth phenotype that correlates with a rapid inhibition of DNA synthesis. Here, using two mutant umuC alleles, one that encodes a UmuC derivative that lacks a detectable DNA polymerase activity (umuC104; D101N) and another that encodes a derivative that is unable to confer cold sensitivity but is proficient for SOS mutagenesis (umuC125; A39V), we show that umuDC-mediated cold sensitivity can be genetically separated from the role of UmuD'(2)C in SOS mutagenesis. Our genetic and biochemical characterizations of UmuC derivatives bearing nested deletions of C-terminal sequences indicate that umuDC-mediated cold sensitivity is not due solely to the single-stranded DNA binding activity of UmuC. Taken together, our analyses suggest that umuDC-mediated cold sensitivity is conferred by an activity of the UmuD(2)C complex and not by the separate actions of the UmuD and UmuC proteins. Finally, we present evidence for structural differences between UmuD and UmuD' in solution, consistent with the notion that these differences are important for the temporal regulation of the two separate physiological roles of the umuDC gene products.

RecA protein of Escherichia coli has a third essential role in SOS mutator activity

The DNA damage-inducible SOS response of Escherichia coli includes an error-prone translesion DNA replication activity responsible for SOS mutagenesis. In certain recA mutant strains, in which the SOS response is expressed constitutively, SOS mutagenesis is manifested as a mutator activity. Like UV mutagenesis, SOS mutator activity requires the products of the umuDC operon and depends on RecA protein for at least two essential activities: facilitating cleavage of LexA repressor to derepress SOS genes and processing UmuD protein to produce a fragment (UmuD') that is active in mutagenesis. To determine whether RecA has an additional role in SOS mutator activity, spontaneous mutability (tryptophan dependence to independence) was measured in a family of nine lexA-defective strains, each having a different recA allele, transformed or not with a plasmid that overproduces either UmuD' alone or both UmuD' and UmuC. The magnitude of SOS mutator activity in these strains, which require neither of the two known roles of RecA protein, was strongly dependent on the particular recA allele that was present. We conclude that UmuD'C does not determine the mutation rate independently of RecA and that RecA has a third essential role in SOS mutator activity.

Ultraviolet mutagenesis and the SOS response in Escherichia coli: a personal perspective.

The study of ultraviolet (UV) mutagenesis in Escherichia coli began with the assumption that genes were likely to be changed at the instant of photon absorption. Over many decades, it became clear that postirradiation cellular activities, including enzymatic DNA repair of UV photo products and error-prone modes of tolerating unrepaired DNA lesions can exert profound influences on the mutagenic outcome of irradiation. Current study focusses on the molecular details of radiation-induced translesion DNA replication as the final event in UV mutagenesis.

The role of exonucleolytic processing and polymerase-DNA association in bypass of lesions during replication in vitro. Significance for SOS-targeted mutagenesis.

The role of exonuclease activity in trans-lesion DNA replication with Escherichia coli DNA polymerase III holoenzyme was investigated. RecA protein inhibited the 3'----5' exonuclease activity of the polymerase 2-fold when assayed in the absence of replication and had no effect on turnover of dNTPs into dNMPs. In contrast, single-stranded DNA-binding protein, which had no effect on the exonuclease activity in the absence of replication, showed a pronounced 7-fold suppression of the 3'----5' exonuclease activity during replication. The excision of incorporated dNMP alpha S residues from DNA by the 3'----5' exonuclease activity of DNA polymerase III holoenzyme was inhibited 10-20-fold; still no increase in bypass of pyrimidine photodimers was observed. Thus, in agreement with our previous results in which the exonuclease activity was inhibited at the protein level (Livneh, Z. (1986) J. Biol. Chem. 261, 9526-9533), inhibition at the DNA level also did not increase bypass of photodimers. Fractionation of the replication mixture after termination of DNA synthesis on a Bio-Gel A-5m column under conditions which favor polymerase-DNA binding yielded a termination complex which could perform turnover of dNTPs into dNMPs. Adding challenge-primed single-stranded DNA to the complex yielded a burst of DNA synthesis which was promoted most likely by DNA polymerase III holoenzyme molecules transferred from the termination complex to the challenge DNA thus demonstrating the instability of the polymerase-DNA association. Addition of a fresh sample of DNA polymerase III holoenzyme to purified termination products, which consist primarily of partially replicated molecules with nascent chains terminated at UV lesions, did not result in any net DNA synthesis as expected. However, reactivation of lesion-terminated primers was achieved by pretreatment with a 3'----5' exonuclease which excised 200 nucleotides or more, generating new 3'-OH termini located away from the UV lesions. When these exonuclease-treated products were subjected to a second round of replication, an increased level of DNA synthesis was observed including additional bypass of photodimers. These results suggest the possibility that 3'----5' exonuclease processing might be required at least transiently during one of the stages of trans-lesion DNA replication, which is believed to be the mechanism of SOS-targeted mutagenesis.